ENRICHMENT AND DETECTION METHODOLOGIES FOR EGFR PHOSPHOPEPTIDES

EGFR 磷酸肽的富集和检测方法

基本信息

批准号：
8170926
负责人：
Vickery E Trinkaus-Randall
金额：
$ 3.71万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8170926
关键词：
Affinity Affinity Chromatography Agonist Antibodies Attenuated Binding Cell Line Cells Computer Retrieval of Information on Scientific Projects Database Data Detection Digestion EGF gene Endothelial Cells Epidermal Growth Factor Receptor Epithelial Cells Exposure to Family suidae Funding Gel Grant Harvest Human Immunoprecipitation Injury Institution Ion Exchange Ions Link Metals Methodology Methods Monitor Mus Mutation Peptide Hydrolases Peptide Mapping Peptides Phase Phenylalanine Phosphopeptides Phosphorylation Phosphorylation Site Play Preparation Procedures Process Protein Tyrosine Kinase Proteins Protocols documentation Receptor Cell Recovery Recruitment Activity Reflex action Research Research Personnel Resources Role Sampling Signal Pathway Site Source Specificity Squamous cell carcinoma Stimulus Techniques Time Tyrosine Tyrosine Phosphorylation United States National Institutes of Health Water base cell growth cell injury corneal epithelium migration phosphatase inhibitor receptor expression response response to injury titanium dioxide tool

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Epidermal growth factor receptor (EGFR) tyrosine kinase plays an important role in regulating cell growth, proliferation, and migration. Differential phosphorylation of specific tyrosine residues in EGFR in response to diverse external stimuli (e.g., exposure to EGF, ATP, cell injury, etc.) serves as the key link between these stimuli and the internal signaling pathways that they activate. EGFR phosphosite-specific antibodies have been used as highly sensitive tools to monitor phosphorylation site occupancy, however, they suffer from a lack of specificity. Recently, highly sensitive mass spectrometric-based detection strategies have been described to characterize the EGFR tyrosine phosphorylation cascade under a variety of conditions. We are exploring the optimization of sample preparation, phosphopeptide enrichment and detection strategies for the study of EGFR phosphopeptides. Human epidermoid carcinoma A431 cells, which over-express EGFR, or the porcine aortic endothelial (PAE) cell line transfected with EGFR expression constructs, or mouse corneal epithelial cells, were grown in culture to confluence, harvested in the presence of a cocktail of protease and phosphatase inhibitors, and EGFR was subjected to immunoprecipitation. Eluted EGFR, or control, commercially-available purified EGFR (derived from A431 cells) was subjected to SDS-PAGE and in-gel digestion with a panel of proteases. Peptides were eluted and subjected to enrichment by reversed-phase, ion exchange, metal ion affinity, or titanium dioxide affinity chromatography. Phosphopeptides were analyzed by MALDI-TOF MS using a variety of matrices and matrix additives in the positive and negative ion modes. We have recovered EGFR from cells in culture with high yield and purity using immunoprecipitation followed by SDS-PAGE. We have explored the use of multiple proteases for in-gel digestion, to target, in particular large tyrosine-containing tryptic peptides that may have been unrepresented in previous MS analyses. Using optimized procedures for recovery from gel, we have subjected the EGFR peptides to various forms of chromatographic enrichment and separation techniques to exploit the differential binding and elution of EGFR phosphopeptides. Using an optimized phosphopeptide recovery and enrichment protocol, followed by MALDI-TOF MS peptide mapping with a Bruker Reflex IV and nanoLC/MSn analysis with a Waters Acuity NanoLC interfaced to an Advion Nanomate and Thermo-Fisher LTQ-Orbitrap MS, we have been able to establish the differential phosphorylation of EGFR in response to injury, and to UTP or EGF stimulation. Phosphorylation of EGFR following UTP stimulation attenuates rapidly compared to direct stimulation of E1PAE cells by EGF. The intensity of EGFR phosphorylation was found to be significantly reduced when cells were stimulated by UTP, as opposed to stimulation by EGF. We observed that Grb2 binding gradually increases over time and reaches its peak at 45 min post-EGF stimulation and attenuates rapidly thereafter. Mutation of tyrosine residues 1068 and 1086 to phenylalanine reduced Grb2 binding when cells were stimulated with UTP, but a smaller decrease was observed when stimulated with EGF. We have also found that neither injury nor UTP, but EGF recruits Grb2 to EGFR. Our data indicates that this is due to the differential phosphorylation of EGFR tyrosine residues that results from stimulation with different agonists. ITRAQ and SILAC methods are now being used to quantify the phosphorylation of EGFR sites and to determine other proteins that serve as interacting parthers in the process.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。表皮生长因子受体（EGFR）酪氨酸激酶在调节细胞生长，增殖和迁移中起重要作用。 EGFR中特定酪氨酸残基的差异磷酸化响应于各种外部刺激（例如，暴露于EGF，ATP，细胞损伤等）是这些刺激与它们激活的内部信号传导途径之间的关键联系。 EGFR磷酸化特异性抗体已被用作高度敏感的工具来监测磷酸化位点的占用率，但是它们缺乏特异性。最近，已经描述了高度敏感的质谱检测策略，以表征EGFR酪氨酸磷酸化级联反应在各种条件下。我们正在探索用于研究EGFR磷酸肽的样品制备，磷酸富集和检测策略的优化。过表达EGFR的人类表皮性癌A431细胞，或用EGFR表达构建体转染的猪主动脉内皮（PAE）细胞系在培养中培养中延长到融合中，在培养物中种植，在蛋白酶和磷酸酶施用的鸡蛋中，对蛋白酶和磷酸酶施加了浓度的鸡尾酒和Egrun dygrun dygrun dygrun andgrun andgrun dygrun dygrun dygrun dygrun andgrun dygrun dygrun andgrun dygrun dygrun。洗脱的EGFR或对照，商业上可用的纯化EGFR（源自A431细胞）进行SDS-PAGE和凝胶消化，并用一组蛋白酶进行凝胶消化。通过反相，离子交换，金属离子亲和力或二氧化钛亲和色谱法对肽洗脱并进行富集。使用MALDI-TOF MS分析磷酸肽，使用阳性和负离子模式中的各种基质和基质添加剂。我们已经使用免疫沉淀，然后是SDS-PAGE从培养物中恢复了EGFR。我们已经探索了多种蛋白酶在凝胶消化中的使用，特别是在先前的MS分析中可能没有代表的大型含酪氨酸的胰蛋白酶肽。使用优化的程序从凝胶中恢复，我们将EGFR肽进行了各种形式的色谱富集和分离技术，以利用EGFR磷酸肽的差异结合和洗脱。使用优化的磷酸肽恢复和富集方案，然后使用Bruker反射IV和Nanolc/MSN分析进行MALDI-TOF MS肽映射，并通过水量nanolc与Adrader-Fisher和Thermo-Fisher LTQ-Orbit MS相连，我们已经在ENANOMATE和THERMO-FISHER LTQ-ORBITRAP MS上进行了互补或EGFFR的响应或EGFR的UTFFR。与通过EGF直接刺激E1PAE细胞相比，UTP刺激后EGFR的磷酸化迅速减弱。当通过UTP刺激细胞而不是通过EGF刺激细胞时，发现EGFR磷酸化的强度显着降低。我们观察到GRB2结合随着时间的推移逐渐增加，并在EGF刺激后45分钟达到其峰值，并在此后迅速减弱。当用UTP刺激细胞时，酪氨酸残基1068和1086与苯丙氨酸的突变减少了GRB2结合，但是当用EGF刺激时，观察到较小的降低。我们还发现既没有伤害也没有UTP，但是EGF将GRB2招募到EGFR。我们的数据表明，这是由于EGFR酪氨酸残基的差异磷酸化引起的，该残基是由不同激动剂刺激引起的。 ITRAQ和SILAC方法现在被用来量化EGFR位点的磷酸化，并确定在此过程中用作相互作用拟南芥的其他蛋白质。